Protease Assay:
-pul 1 ml of vibrio tubiashii culture in centrifuge tube and centrifuge at 4C for 10 mins at 13,000 rpm
-transfer 100 ul of the supernatant to a new tube
-add 400 ul of 1% azocasein
-incubate for 30 min at 37C in a heat block
-terminate the rxn with the addition of 600 ul of 10% trichloric acetic acid (TCA)
-incubate on ice for 30 min
-add 200 ul of 1.8 N NaOH to a new microcentrifuge tube
-add 800 ul of the supernatant from the experimental tube to the new tube with NaOH

the Azocasein Protease Assay is used to identify pathogenic vibrio sp.

a positive protease result will be indicated by a magical orange color change
a negative test result will be indicated by no color change and the solution will remain clear/ clearish

-my experimental tube turned bright orange instantly with the adition of the supernatant to the NaOH! magic!
-a positive result in this experiment suggests that V. tubiashii (or any other tested vibrio sp.) has protease and the ability to breakdown proteins.
-the specific protease tested for is metalloprotease
-had i not known what sp. of vibrio i had i could perform the agglutination test we learned yesterday that is specific for v. tubiashii to test/confirm the unkown vibrio pathogen as v. tubiashii

Differential Display:
-this experiment was performed as a class with infected and non infected sea fan samples sent from Cornell University to look for differences in gene expression between infected and healthy sea fans
-differential display uses random primers to look at differences in in your amplified cDNA on a gel
steps:
-extract DNA
-clean RNA and normalize to same concentration
-make cDNA and adapter (1 specific primer)
-do PCR (1 specific primer and 1 random primer)
... this is as far as we went in lab today, tmr we will...
-run a gel> look for differences in the banding patterns to see if things are differentially expressed
... this is as far as we will take this in class
if you were doing this on your own and wanted to go farther or get more information you could:
-excise the band of interest> clean> sequence
-confirm with qPCR

August 2, 2010
Lab 13

Serum Agglutination Test:
-used a 2 well depression slide: 1=control 2=experimental
-control: added 50 ul sterile seawater and 25 ul of culture (marine broth + vibrio tubiashi)
-exp: added 25 ul sterile seawater, 25 ul of culture and 25 ul polyclonal antibody
-both wells were gently mixed with a pippette and allowed to incubate at RT for 5 mins
-examination with a compound microscope at 10x allowed for agglutination comparison between the 2 wells
-the experimental well had obvious clumps/ obvious agglutination
-the antiobody stuck to the antigens on the vibrio tubiashi and clumps the bacteria together allowing you to see agglutination
-the antibody used was specific for vibrio tubiashii

Oyster Larvae project:
-changed water in each of the 8 tanks
-sampled for RNA and mortality counts
-Neutral Red dye did not work properly so we re-sampled and performed counts for alive, swimming and dead on unstained larvae with dissection scopes

July 31, 2010
Lab 12

Oyster Larvae Project:
-we changed the water in each of the 8 larvae holding tanks by:
removing the tanks filtering out the larvae through a 70um mesh filter
took two larval samples from each tank: one for mortality counts (alive, swimming, moribund and dead) to be stained with a vital stain, Neutral Red (red=alive, pink=moribund, white/clear=dead) and another sample of ~1000 larvae for RNA samples that we put into RNAlater in tubes corresponding to each holding tank
we then put the larvae back into the tanks with fresh fileterd sea water
-we fed the larvae algae grown in the lab
-we innoculated the larvae tanks with v. tubiashii in varying concentrations

Adventure to Cattle Point, not the poo site:
-Marie, Kathy and I went to the lighthouse site at Cattle point and collected 15 anthopleura elegantissima from the intertidal
-we removed the anemones off rocks in and out of the water with a metal spatula
-we placed the anemones into glass dishes back at the labs and put them in a 15C tank to acclimate

July 30, 2010
Lab 11

Western Blot Round 2:
-we attempted the western blot again following protocol
-this time we used anemone tentacles, anemone acontia, an oyster sample from steven and a barnacle sample we collected
-we loaded 50ul of each sample into our gel wells
-unfortunately the anemone and oyster samples did not give us any bands, however we did get some magic from the barnacle sample, hooray!
-in thinking about our group project to investigate heat stress in anemones we decided we may need to use a more sensitive method than the western to detect the heat shock proteins, maybe QPCR

Oyster Larvae Project:
-we changed the water in each of the 8 larvae holding tanks by:
removing the tanks filtering out the larvae through a 70um mesh filter
took two larval samples from each tank: one for mortality counts (alive, swimming, moribund and dead) to be stained with a vital stain, Neutral Red (red=alive, pink=moribund, white/clear=dead) and another sample of ~1000 larvae for RNA samples that we put into RNAlater in tubes corresponding to each holding tank
we then put the larvae back into the tanks with fresh fileterd sea water
-we fed the larvae with algae grown in the lab

Field Trip to Argyle:
-we heard that there were infected/sick Molpalia chiton, hairy chitons at Argyle
-we went to Arglye and searched the shallow water between the two bays for diseased chiton
-we collected several chitons healthy and diseased
-healthy chiton were firmly attached to rocks
-diseased chiton were collected if they were not firmaly attached to rocks after we poked them or if we found them upside down (foot side up)
-healthy and diseased chitons were placed in separate bags and taken back to lab for examination, swabbing and plating

July 29,2010
Lab 10

Protein Gel:
-we looked at our protein gels from yesterday to decide on an appropriate usable concentration
-We decided to run another gel for a western blot where we transfer the image from the gel onto a membrane
-we ran the other gel for 45 min at 150 volts

Western Blot:
-we set up the western blot to look at heat shock proteins using an anti-HSP70 antibody and a kit from invitrogen: the WesternBreeze Chromogenic Western Blot Immunodetection Kit
-the anti-HSP70 antibody works well on oysters
-unfortunately no bands transferred to our filter paper (it would have been a good idea to run a ladder on our gel so that we could troubleshoot what went wrong because the ladder will always transfer
-no bands= no magic
-to see if any proteins had transferred to the membrane we stained it with a Ponceau stain which will stain proteins on the membrane red/pink; no proteins stained
-we followed the protocol correctly however reasons for no bands may have been: improper/no binding of the anti-HSP70 on the anemones (this antibody works well for oysters... maybe not for anemones), we borrowed the antibody from another lab and maybe we need the fresh stuff, it is possible but not likely that the amount of protein we used was not enough or maybe the heat shock proteins were not expressed because we did not heat stress the organisms enough (although background staining is usually observed)
-looking through the literature we found an article Snyder & Rossi_2004_Stress Protein (HSP70 family) expression in intertidal benthic organisms: the benthic organisms: the example of Anthopleura elegantissima (Cnidaria: Anthozoa)_Sci. mar., 68 (1) : 155-162, that was investigating heat stress in anemones and they stated that most of the protein expression was seen in the tentacles (basically all of the action happens in the tentacles), it is likely that we diluted the HSP's in our samples by including much of the body in our sample

Oyster dissection:
-Ostrea edulis was ordered for the class to investigate the presence of Bonamia
-we opened the animal by prying the valves open with a butter knife and severing the adductor muscle
-once inside we identified the gills, palps, fast and slow twitch muscles, heart and digestive gland.
-we opened the pericardial cavity and found the three chambered heart: an off white ventricle with two dark brown auricles
-we removed the heart and separated the ventricle from the auricles
-we attempted to remove blood from the heart with a syringe but were unable to get any fluid so we blotted the outside and inside of the heart onto a glass slide
-we cut off a piece of the digestive gland and also blotted it on a slide
-we placed both slides in methanl for 1 minute to fix the cells and adhere them onto the slide
-next we placed the slides in Giemsa stain for 2 mins, followed by a water wash
-we observed the slides with LM and the blood appeared normal
-no signs of Bonamia, blood cells looked good

July 28, 2010
Lab 9

ISH continued:
-today we completed our ISH for visualization of WS-RLP on our abalone tissue slides by completing the final detection steps (7-10) from the protocol: http://bio533.wikispaces.com/Lab_Withering
-no one in our has success with the ISH
-no one could detect staining with the probe to see any rickettsia-some slides may have been been lightly stained, but nothing confirmatory
-if the stain had worked properly we would have been able to visualize the tissue with light brown staining and the rickettsia cells/ inclusions would wave appeared dark brown/purple/black

ISH and H&E slides:
-we looked at slides in which ISH had been successful in the past
-we saw the rickettsia clearly stained at dark brown inclusions
-we looked at H&E slides showing the 3 strains of rickettsia: classic, new and stippled
-the inclusions were composed of rickettsial colonies
-the new strain can be identified by larger and darker inclusions
- the classic strain is lighter in color and makes smaller inclusions
-the stippled strain can be differentiated from the others due to darker spots within the inclusions (stippled appearance)

Protein work: SDS-PAGE gel:
-we ran a protein gel on heat shocked and control Anthopleura anemones
-the heat shocked organisms were placed in 25C water overnight and 29C water this morning
-the control orgs were kept at RT
-we extracted protein form the animals: 6 experimental and 6 control by making longitudinal cuts through the orgs and collecting tissue samples weighing ~ 0.025g (my sample weight: 0.02g)
-once the tissue was collected the samples were ground up and lysed
-following protein extraction the samples were mixed with a loading dye and each loaded into 3 separate wells on a polyacrylamide gel in 3 different amounts: 10ul, 30ul and 50ul
-the protocol for protein extraction (in the protein extraction section): http://bio533.wikispaces.com/Lab_Proteomic
-the protocol for loading the gel (in the protein gel protocol section): http://bio533.wikispaces.com/Lab_Proteomic
the protocol was augmented in step 3 where we used 25ul of all reagents
-the gel was run for 45 mins at 150 volts
-the gel was stained in Coomasie dye for 5 mins and destained with acetic acid
-the gel will give us information on the quantity and quality of protein in our samples

July 27, 2010
Lab 8

ISH continued:
- we continued working with our abalone WS-RLP slides to visualize the disease.
-we continued working off the ISH protocol: http://bio533.wikispaces.com/Lab_Withering-today we completed the following steps:
stringency washes
steps 1-6 of detection
-we were unable to visualize the stain after 15 min so we let the slides go overnight to ensure detection
July 26, 2010
Lab 7

ISH:
-today we worked with abalone histology slides and began in situ hybridization to visualize withering syndrome (RLP) of the abalone tissue
-we used the Antonio (2001) protocol: http://bio533.wikispaces.com/Lab_Withering.
-today we began the process by completing the following steps:
tissue deparaffinization
tissue permeabilization
hybridization

July 24, 2010
Lab 6

Armina Plates:
-put plates in -4C to stop growth

PCR and QPCR analysis:PCR
-in our PCR gel analysis we recovered bacterial DNA bands
-we had a slight primer dimer and our negative control was not clean
-because our negative control showed contamination the best thing to do would be to re-do the PCRQPCR
-QPCR is good for DNA and RNA and identifying the quantity of a pathogen and for looking at gene expression
-SyBr green binds to the dsDNA, (all dsDNA: which allows the potential fluorescence from non-specific products) it is not the most specific QPCR chemistry but it has advantages that it only requires primers and you can get a melting curve
-the melting curve analysis plots the negative rate of change of fluorescence against temperature
-in our QPCR assay we want only one specific product to amplify
-for a good assay you need: quality RNA, no DNA, good replication and an appropriate normalizing target
-the dissociation curve for our actin reference gene amplified at ~85 and our gene of interest cjunk amplified at ~80
-there was some contamination in our QPCR
-using the software data from our QPCR we will analyze by obtain our CT values, using a formula to get an
arbitrary expression value=10∧(-(0.3012*CT)+11.434)
-gene expression:cjunk
-0.525
-yes contaminationactin
-5.14
-yes contamination
now we must normalize
to normalize the data divide the cjunk exp. by the actin exp.= 0.102
by normalizing the data you can compare the samples
our sample size is small so it is hard to make many conclusions but we have a good start even though our data is not significant

July 23, 2010
Lab 5

48 hour Plate observations:
-Marine agar L
-L1: Q1 lots of growth, Q2 6 colonies, Q3 bacteria from Q1 due to condensation in the plate
-L2: Q1 lots of growth, Q2 14 colonies, no bacteria in Q3 or 4
-L3:Q1 lots of growth, Q2 11 colonies, no colonies in Q3 or 4
-colonies still appeared small, round, white, mucoid
-TCBS agar L
-L1: some single colonies in Q1, condensation had spread bacteria in Q2, 3, 4
-L2: 20+ colonies in Q1 along with some blobby growth, Q2 no growth, Q3 some growth from Q1 due to condensation, Q4 lots of growth due to condensation from Q1
-L3: Q1 had 4 colonies, lots of growth in Q2, some spread into Q3 and 4 from condensation from Q1
-all plates had turned a yellow/ orange color since yesterday due to bacterial fermentation of sucrose
-some colonies appeared orange, shiny, mucoid, round with complete margins and convex
-some colonies appeared green, shiny, mucoid

Plate observations:
-observed the marine agar and TCBS plates that we streaked yesterday with infected Armina lesions
-growth was observed on all lesion plates with both types of agar
-every plate had lots of growth in quadrant 1 the initial swab
-for the marine agar bacterial growth was observed in Q1 and single colonies were produced in Q2, no growth was seen in Q3 and 4
-for the TCBS agar plates bacterial growth was observed in Q1 and single colonies were produced in Q2, some growth was seen in Q3 and 4 however it looks like the growth from Q1 spread into these quadrants due to water from rinsing the loop or condensation from the plate
-the TCBS agar produced growth of different types of vibrio due to color changes: most of the colonies on the TCBS plates were yellow indicating vibrio colonies capable of fermenting sucrose, some colonies were green indicating vibrio colonies unable to ferment sucrose
-on all of the plates most growth was found in Q1 from the initial swab
-the single colonies that grew were smooth/round colonies with smooth/ complete margins
-small, white, round colonies on the marine agar plates
- small yellow and green colonies on the TCBS agar plates
-allow the plates to grow another day to see if we can get more growth

Plating
-obtained heathy and diseased Armina from the Neuroethology class which were collected near Tacoma, WA
-healthy animals are an orange tan color with white stripes running longitudinally down their backs
-sick animals have lesions (melting of the epidermis) on the epidermis to varying degrees and exhibit behavioral differences
-the class split into groups to investigate the healthy and unhealthy animals taking samples from lesion and non-lesion areas
-our group worked on a sick animal ( from the healthy tank with multiple lesions: DIS-HEALTH-1
-swabbed epidermis and streaked onto TCBS and marine agar plates in triplicate
-lesion plates L 1,2,3; non lesion plates NL 1,2,3
-used a sterile swab to get the epidermal sample and streaked this into the 1st of 4 quadrants on the plates
-used a sterile plastic loop to streak into the following quadrants 2,3,4; rinsing the loop in bleach and tap water each time
-plates were parafilmed and left out for later observation of bacterial growth
-squash mounts of the lesion and non lesion scrapings were observed with a compound scope and we observed amoeba-like parasites in the sick animals; fewer cells came off of the healthy animals
-these were allowed to air dry and later gram stained allowing for observation of numerous pink (gram-) rods, possibly vibrio bacteria
-gram stain protocal can be found at: http://bio533.wikispaces.com/Lab_Littorina

Littorina dissection:
-dissected two species of Littorina: L. scutulata and L. sikana collected form Cattle Point
methods:
1) collected organism, cracked shell with a vice, removed shell
2) cut off head, digestive gland and operculum
3) placed remaining body tissue in a RNase free labeled tube and put in the -80C freezer in the dry ice box for RNA extraction
4) cut off a small piece of foot from the operculum for DNA analysis, placed in labeled tube and put in the -20C freezer
5) observed digestive gland under the dissecting scope to check for parasites
- I dissected and processed 3 L.sikana: organisims appeared healthy with no obvious signs of infection
- I dissected and processed 3 L. scutulata: organisms appeared healthy with no obvious signs of infection

- other lab mates found trematode infections of varying degrees upon observation of the digestive glands in both species
- the trematodes differed and some interesting parasitic features included: suckers, eyespots, tail spikes

Lab 1
Invert Anatomy

Today I dissected Cucumaria, a sea cucumber.
"Tiny" the sea cucumber was very large with a long, cucumber shaped body.
Immersed in seawater we (Matthew and I) made a longitudinal cut from anus to mouth (posterior to anterior) through the thick body wall to open the body cavity.
We pinned the animal open and found beautiful guts.
Behind the mouth we could see the calcareous ring, below that hung a brown esophagus followed by a small, tan stomach and a long intestine leading to the anus.
Long, orange, stringy, spaghetti like gonads could be seen in the body cavity around the stomach/ anterior intestine.
The major respiratory organ, the respiratory tree, could be seen as a lovely, clear, fragile system of branching tubes hanging off of the posterior intestine.